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Title page for ETD etd-03212009-152926


Type of Document Dissertation
Author Mavencamp, Terri Lynn
URN etd-03212009-152926
Title Design, Synthesis and Biological Evaluation of a Family of Excitatory Amino Acid Transporter 3 (EAAT3) Preferring Inhibitors.
Degree Doctor of Philosophy
Department Department of Biomedical & Pharm. Sciences
Advisory Committee
Advisor Name Title
Christopher S Esslinger Committee Chair
Diana I Lurie Committee Member
Mark L Grimes Committee Member
Michael P Kavanaugh Committee Member
Richard J Bridges Committee Member
Keywords
  • Benzyl Aspartate
  • EAAT3
  • Glutamate
  • Pharmacology
  • Transporters
Date of Defense 2008-01-01
Availability unrestricted
Abstract

This work describes the synthesis and initial characterization of the biological activity of a family of EAAT3 preferring inhibitors, L-β-benzyl aspartate (L-β-BA) and L-β-BA derivatives. L-β-BA and derivatives were initially synthesized in an approximate 2:1 ratio of diasteromers (threo:erythro), using base promoted enolate addition. Kinetic analysis of 3H-D-aspartate uptake into C17.2 cells expressing the hEAATs demonstrated that L-threo-β-BA is the more potent diastereomer (Ki values of 9 然 for EAAT1, 10.0 然 for EAAT2 and 0.8 然 for EAAT3), acts competitively, and exhibits a 10-fold preference for EAAT3 compared to EAAT1 and EAAT2. Electrophysiological recordings of EAAT-mediated currents in Xenopus oocytes further identified L-β-BA as a non-substrate inhibitor. Derivatives of L-β-BA were prepared and characterized for the ability to inhibit 3H-D-aspartate uptake into hEAAT1-3 expressing C17.2 cells. Computational modeling and analysis of structure activity data suggest the area the aromatic moiety of L-β-BA derivatives probe is 1) 3-dimentionally confined, 2) more tolerant of substitutions at the 3 and 5 positions than the 4 position, 3) at least partially distinct from the area probed by L-TBOA and 4) more accessible in the EAAT3 protein than EAAT1 and EAAT2. Computational modeling supports the pharmacological data and lends insight into the selectivity observed with L-β-BA derivatives. Docking studies suggest that H-bonding interactions of L-β-BA derivatives with key residues in the binding site position L-β-BA analogues in a unique manner that is better tolerated in the EAAT3 protein than in the EAAT1 and EAAT2 proteins.

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